Membrane-bound Substrates Research Articles

Phosphatidate phosphatase Pah1 contains a novel RP domain that regulates its phosphorylation and function in yeast lipid synthesis

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The enzyme controls whether cells utilize PA to produce membrane phospholipids or the major storage lipid triacylglycerol. PA levels, which are regulated by the enzyme reaction, also control the expression of UASINO-containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Pah1 function is largely controlled by its cellular location, which is mediated by phosphorylation and dephosphorylation. Multiple phosphorylations sequester Pah1 in the cytosol and protect it from 20S proteasome-mediated degradation. The endoplasmic reticulum-associated Nem1-Spo7 phosphatase complex recruits and dephosphorylates Pah1 allowing the enzyme to associate with and dephosphorylate its membrane-bound substrate PA. Pah1 contains domains/regions that include the N-LIP and haloacid dehalogenase-like catalytic domains, N-terminal amphipathic helix for membrane binding, C-terminal acidic tail for Nem1-Spo7 interaction, and a conserved tryptophan within the WRDPLVDID domain required for enzyme function. Through bioinformatics, molecular genetics, and biochemical approaches, we identified a novel RP (regulation of phosphorylation) domain that regulates the phosphorylation state of Pah1. We showed that the ΔRP mutation results in a 57% reduction in the endogenous phosphorylation of the enzyme (primarily at Ser-511, Ser-602, and Ser-773/Ser-774), an increase in membrane association and PA phosphatase activity, but reduced cellular abundance. This work not only identifies a novel regulatory domain within Pah1 but emphasizes the importance of the phosphorylation-based regulation of Pah1 abundance, location, and function in yeast lipid synthesis.

Targeting membrane-bound bacterial cell wall precursors: a tried and true antibiotic strategy in nature and the clinic.

Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.

Negative evidence for a role of APH1B T27I variant in Alzheimer’s disease

γ-secretase is a macromolecular complex that catalyzes intramembranous hydrolysis of more than 100 membrane-bound substrates. The complex is composed of presenilin (PS1 or PS2), anterior pharynx defect-1 (APH-1), nicastrin (NCT) and PEN-2 and early-onset; autosomal dominant forms of Alzheimer's disease (AD) are caused by inheritance of mutations of PS. No mutations in genes encoding NCT, or PEN-2 have been identified to date that cause AD. In this regard, a large genetic meta-analysis of four cohorts consisting of more than 600 000 individuals identified a common missense variant, rs117618017 in the APH1B gene that results in a T27I mutation, as a novel genome-wide significant locus. In order to confirm the findings that rs117618017 is associated with risk of AD, we performed a genetic screen from deep whole genome sequencing of the large NIMH family-based Alzheimer's Disease (AD) dataset. In parallel, we sought to uncover potential molecular mechanism(s) by which APH-1B T27I might be associated with AD by generating stable HEK293 cell lines, wherein endogenous APH-1A and APH-1B expression was silenced and into which either the wild type APH-1B or the APH-1B T27I variant was stably expressed. We then tested the impact of expressing either the wild type APH-1B or the APH-1B T27I variant on γ-secretase processing of human APP, the murine Notch derivative mNΔE and human neuregulin-1. We now report that we fail to confirm the association of rs1047552 with AD in our cohort and that cells expressing the APH-1B T27I variant show no discernable impact on the γ-secretase processing of established substrates compared with cells expressing wild-type APH-1B.

ADAM10 mediates malignant pleural mesothelioma invasiveness

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options and treatment efficiency. Even if the latency period between asbestos exposure, the main risk factor, and mesothelioma development is very long, the local invasion of mesothelioma is very rapid leading to a mean survival of one year after diagnosis. ADAM10 (A Disintegrin And Metalloprotease) sheddase targets membrane-bound substrates and its overexpression is associated with progression in several cancers. However, nothing is known about ADAM10 implication in MPM. In this study, we demonstrated higher ADAM10 expression levels in human MPM as compared to control pleural samples and in human MPM cell line. This ADAM10 overexpression was also observed in murine MPM samples. Two mouse mesothelioma cell lines were used in this study including one primary cell line obtained by repeated asbestos fibre injections. We show, in vitro, that ADAM10 targeting through shRNA and pharmacological (GI254023X) approaches reduced drastically mesothelioma cell migration and invasion, as well as for human mesothelioma cells treated with siRNA targeting ADAM10. Moreover, ADAM10 downregulation in murine mesothelioma cells significantly impairs MPM progression in vivo after intrapleural cell injection. We also demonstrate that ADAM10 sheddase downregulation decreases the production of a soluble N-cadherin fragment through membrane N-cadherin, which stimulated mesothelioma cell migration. Taken together, we demonstrate that ADAM10 is overexpressed in MPM and takes part to MPM progression through the generation of N-cadherin fragment that stimulates mesothelioma cell migration. ADAM10 inhibition is worth considering as a therapeutic perspective in mesothelioma context.

Transmembrane Protease TMPRSS11B Promotes Lung Cancer Growth by Enhancing Lactate Export and Glycolytic Metabolism

SUMMARYPathways underlying metabolic reprogramming in cancer remain incompletely understood. We identify the transmembrane serine protease TMPRSS11B as a gene that promotes transformation of immortalized human bronchial epithelial cells (HBECs). TMPRSS11B is upregulated in human lung squamous cell carcinomas (LSCCs), and high expression is associated with poor survival of non-small cell lung cancer patients. TMPRSS11B inhibition in human LSCCs reduces transformation and tumor growth. Given that TMPRSS11B harbors an extracellular (EC) protease domain, we hypothesized that catalysis of a membrane-bound substrate modulates tumor progression. Interrogation of a set of soluble receptors revealed that TMPRSS11B promotes solubilization of Basigin, an obligate chaperone of the lactate monocarboxylate transporter MCT4. Basigin release mediated by TMPRSS11B enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. These findings establish an oncogenic role for TMPRSS11B and provide support for the development of therapies that target this enzyme at the surface of cancer cells.

Optimizing the localization of astaxanthin enzymes for improved productivity

BackgroundOne important metabolic engineering strategy is to localize the enzymes close to their substrates for improved catalytic efficiency. However, localization configurations become more complex the greater the number of enzymes and substrates is involved. Indeed, optimizing synthetic pathways by localizing multiple enzymes remains a challenge. Terpenes are one of the most valuable and abundant natural product groups. Phytoene, lycopene and β-carotene serve as common intermediates for the synthesis of many carotenoids and derivative compounds, which are hydrophobic long-chain terpenoids, insoluble in water and usually accumulate in membrane compartments.ResultsWhile β-ionone synthesis by β-carotene cleavage dioxygenase PhCCD1 and astaxanthin synthesis by β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) differ in complexity (single and multiple step pathways), the productivity of both pathways benefited from controlling enzyme localization to the E. coli cell membrane via a GlpF protein fusion. Especially, the astaxanthin synthesis pathway comprises both CrtW and CrtZ, which perform four interchangeable reactions initiated from β-carotene. Up to four localization strategies of CrtW and CrtZ were exhaustively discussed in this work, and the optimal positioning strategy was achieved. CrtW and CrtZ were linked using a flexible linker and localized to the membrane via a GlpF protein fusion. Enzymes in the optimal localization configuration allowed a 215.4% astaxanthin production increase.ConclusionsThis work exploits a localization situation involving membrane-bound substrates, intermediates and multiple enzymes for the first time, and provides a workable positioning strategy to solve problems in similar circumstances.

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.

Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities

γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

DPP-4 inhibitors-renoprotection in diabetic nephropathy?

There is no doubt that rates of chronic kidney disease are escalating and that this rise is the main contributor to the increasing prevalence of diabetic nephropathy. It is also clear that the increase in kidney failure continues despite tight blood glucose and blood pressure control, as well as renin-angiotensin system blockade. In response, research into novel therapies to treat diabetic nephropathy is expanding, but to date nothing has translated into clinical use. The dipeptidyl peptidase-4 (DPP-4) inhibitors are oral, weight-neutral hypoglycemic drugs used to treat patients with type 2 diabetes. DPP-4 cleaves polypeptides with a proline/alanine in the penultimate position at the amino-terminal position. Hence, the net physiologic effect is a complex interplay between the resulting substrate/product profile in a particular disease milieu (rather than a specific signaling pathway). Cleaved substrates may either be activated, inactivated, or bear no functional relevance. DPP-4 inhibitors lower blood glucose levels by raising the half-life of short-lived endogenous incretins, such as GLP-1 and glucose-dependent insulinotropic polypeptide. However, the ability of DPP-4 to cleave a host of additional membrane-bound substrates that exert nonenzymatic properties by interacting or colocalizing with other membrane proteins/receptors suggests it may be a novel and …

Role of cryo-ET in membrane bioenergetics research

To truly understand bioenergetic processes such as ATP synthesis, membrane-bound substrate transport or flagellar rotation, systems need to be analysed in a cellular context. Cryo-ET (cryo-electron tomography) is an essential part of this process, as it is currently the only technique which can directly determine the spatial organization of proteins at the level of both the cell and the individual protein complexes. The need to assess bioenergetic processes at a cellular level is becoming more and more apparent with the increasing interest in mitochondrial diseases. In recent years, cryo-ET has contributed significantly to our understanding of the molecular organization of mitochondria and chloroplasts. The present mini-review first describes the technique of cryo-ET and then discusses its role in membrane bioenergetics specifically in chloroplasts and mitochondrial research.

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

CAMP-Dependent Protein Kinase A Selects the Excited State of the Membrane Substrate Phospholamban

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca2+-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.

A Myristoyl/Phosphoserine Switch Controls cAMP-Dependent Protein Kinase Association to Membranes

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.

Insights into Substrate Gating in H. influenzae Rhomboid

Rhomboids are a remarkable class of serine proteases that are embedded in lipid membranes. These membrane-bound enzymes play key roles in cellular signaling events, and disruptions in these events can result in numerous disease pathologies, including hereditary blindness, type 2 diabetes, Parkinson's disease, and epithelial cancers. Recent crystal structures of rhomboids from Escherichia coli have focused on how membrane-bound substrates gain access to a buried active site. In E. coli, it has been shown that movements of loop 5, with smaller movements in helix 5 and loop 4, act as substrate gate, facilitating inhibitor access to rhomboid catalytic residues. Herein we present a new structure of the Haemophilus influenzae rhomboid hiGlpG, which reveals disorder in loop 5, helix 5, and loop 4, indicating that, together, they represent mobile elements of the substrate gate. Substrate cleavage assays by hiGlpG with amino acid substitutions in these mobile regions demonstrate that the flexibilities of both loop 5 and helix 5 are important for access of the substrates to the catalytic residues. Mutagenesis indicates that less mobility by loop 4 is required for substrate cleavage. A reexamination of the reaction mechanism of rhomboid substrates, whereby cleavage of the scissile bond occurs on the si-face of the peptide bond, is discussed.

Role of N-Myristoylation of Camp-Dependent Protein Kinase a in Recognition and Phosphorylation of Membrane-Bound Substrates

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C), controls the activation and deactivation of over 100 unique cytosolic and membrane bound substrates. Nearly all the PKA-C in mammals is subjected to myristoylation and myristoylation can be dependent on cell/tissue type. Although the function of myristoylation is not known, it has been hypothesized to mediate PKA-C/membrane and PKA-C/membrane protein interactions. Here, we developed an in vitro myristoylation method to obtain homogeneously myristoylated PKA-C (myrPKA-C) in quantities suitable for NMR studies. Using NMR spectroscopy, the interactions of membrane mimicking isotropic bicelles (DMPC/DHPC) with 15N-labeled myrPKA-C or 15N-labeled non-myristoylated PKA-C was monitored by [1H-15N]-TROSY-HSQC spectroscopy. Significant chemical shift perturbations were detected for the N-terminus and the myristate pocket of myrPKA-C, while both perturbations and line broadening was detected in remote regions far away from the myristoyl binding pocket. Such changes were not observed for the non-myristoylated form, suggesting that myristoylation drives the enzyme towards the membrane and may act allosterically on the enzyme. Membrane localization was further supported by intrinsic tryptophan fluorescence measurements which showed drastically different patterns between myrPKA-C and PKA-C. Finally, the insertion of the myristate group into lipid membranes was confirmed using 2H solid-state NMR spectroscopy on myrPKA-C with a deuterated myristoyl group. We propose that the myristoyl group of myrPKA-C steers the enzyme towards the membrane, even in the absence of its regulatory subunit or A-kinase anchoring proteins (AKAPs). These results provide an additional mechanism for PKA localization and recognition of membrane bound substrates. Preliminary data on PKA recognition of the integral membrane protein, phospholamban, are also presented.

Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β-related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface-targeted fluorescent biosensor (cell surface-linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

Structure of the bacterial teichoic acid polymerase TagF provides insights into membrane association and catalysis

Teichoic acid polymers are composed of polyol-phosphate units and form a major component of Gram-positive bacterial cell walls. These anionic compounds perform a multitude of important roles in bacteria and are synthesized by monotopic membrane proteins of the TagF polymerase family. We have determined the structure of Staphylococcus epidermidis TagF to 2.7-A resolution from a construct that includes both the membrane-targeting region and the glycerol-phosphate polymerase domains. TagF possesses a helical region for interaction with the lipid bilayer, placing the active site at a suitable distance for access to the membrane-bound substrate. Characterization of active-site residue variants and analysis of a CDP-glycerol substrate complex suggest a mechanism for polymer synthesis. With the importance of teichoic acid in Gram-positive physiology, this elucidation of the molecular details of TagF function provides a critical new target in the development of novel anti-infectives.

A Conserved Membrane Attachment Site in α-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of alpha-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.

ADAM10 as a Therapeutic Target for Cancer and Inflammation

Both cancer and chronic inflammatory diseases are often marked by homeostatic signal transduction pathways run amok. Cleavage of membrane-bound substrates by extracellular metalloproteinases is frequently the rate limiting step in activating many of these pathways, resulting either in liberation of active ligands (shedding) or initiating further processing into bioactive cytoplasmic domains (regulated intramembrane proteolysis or RIP). ADAM10 is a member of the ADAM (A Disintegrin And Metalloproteinase) family of transmembrane metalloproteinases implicated in the RIPing and shedding of dozens of substrates that drive cancer progression and inflammatory disease, including Notch, E-cadherin, EGF, ErbB2 and inflammatory cytokines. ADAM10's emerging role as a significant contributor to these pathologies has led to intense interest in it as a potential drug target for disease treatment. Here we discuss some of the established functions of ADAM10 and the implications of its inhibition in disease progression.

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

γ-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing amyloid precursor protein or Notch fragments. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or β-galactosidase assay. We screened and evaluated γ-secretase mutants using this reconstitution system in yeast, which does not possess endogenous γ-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the β-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid β peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of γ-secretase.